This proposal focuses on the development, regulation and discovery of naturally occurring antimicrobial proteins in the epididymis, highlighting the defensins and cathelicidins. Natural antimicrobial proteins are part of the innate immune system, and they likely protect the reproductive tract from invasion by pathogenic microbes thus helping to prevent diseases such as epididymitis. The first aim is to identify novel antimicrobial epididymal proteins in epididymal luminal fluid and to test their biological activities. The reproductive organs produce a variety of antimicrobial substances and it is likely that some of these natural antibiotics remain to be characterized. A step-wise approach will be used to test for antibacterial activity in luminal fluid from different regions of the epididymis, and subsequently to identify, characterize, and analyze the specificities of antimicrobial proteins. The second aim is to determine the pattern of expression of mRNA and protein for selected defensins and cathelicidin during development of the rat epididymis. These studies will determine the temporal onset of expression, quantify changes in expression levels during development, and localize expression to specific epithelial cell types. The third aim is to determine factors regulating expression of antimicrobial proteins in the epididymis by testing the effects of androgens, luminal fluid, exposure to bacterial products, and obstruction on expression of selected genes. Methods include detection of antimicrobial proteins by a gel overlay method and other bacteriologic assays, northern and western analyses, real time PCR, in situ hybridization, and immunohistochemistry, 2-D gel electrophoresis, microsequencing by mass spectrometry, and standard approaches of molecular biology. A set of antimicrobial proteins known to be present in the rat epididymis will be studied in aims 2 & 3: rat beta defensins 1 and 2 (RBD-1 and RBD-2), Bin1b, a cathelicidin (rCRAMP), and the defensin-like molecule E-3. The proposed studies will increase our knowledge of innate antimicrobial proteins in the male reproductive system, and they also present the opportunity for discovery and characterization of new antibiotic agents.